Methods and reagents for G -65 -globin typing

ABSTRACT

Methods and reagents for determining an individual&#39;s genotype at the  G  γ-globin locus with respect to a set of three alleles using sequence-specific oligonucleotide probes enable one to type samples from a variety of sources, including samples comprising RNA or cDNA templates, and can be applied to nucleic acids in which a target region spanning the polymorphism has first been amplified. This three-allele system facilitates typing tissue for determining individual identity and has application in the field of forensic science.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides methods and reagents for determining anindividual's genotype at the ^(G) γ-globin locus with respect to a setof three alleles using sequence-specific oligonucleotide probes. Theinvention enables one to type samples from a variety of sources,including samples comprising RNA or cDNA templates, and can be appliedto amplified nucleic acids containing the target polymorphic region. Thethree-allele system provides substantially more information than theprior art bi-allelic system based on the polymorphic HindIII site. Thepresent typing system facilitates typing tissue for determiningindividual identity and is especially useful in the field of forensicscience.

2. Description of Related Art

The β-related globin proteins are encoded by genes on the short arm ofhuman chromosome 11. The ^(G) γ-globin and ^(A) γglobin genesrespectively code for the ^(G) γ- and ^(A) γ-polypeptide chains of α₂^(G) γ₂ and α₂ ^(A) γ₂, the major species of fetal hemoglobin producedby the fetal liver. The β- and δ-globin genes are activated aroundbirth, while the two γ-globin genes are inactivated. The two adulthemoglobins, produced in the bone marrow, are α₂ β₂ and α₂ δ₂. Thenucleotide sequences of the ^(G) γ-globin and ^(A) γ-globin genes werefirst described by Slightom et al., 1980, Cell 21:627-638. Each geneconsists of three exons and two intervening sequences (IVS).

The ^(G) γ-globin and ^(A) γ-globin genes have been classified byvariant patterns of restriction endonuclease cleavage site number andlocation. In particular, a HindIII site polymorphism has been discovered(Jeffreys, 1979, Cell 18:1-10) near the 3' end of the second IVS.

Nucleotide sequence variability corresponding to the HindIII sitepolymorphism has been observed in the study of gene conversion in thetwo fetal globin genes (Shiokawa et al., 1989, J. Biochem. 105:184-189,incorporated herein by reference). Probes for the sequencescorresponding to the HindIII polymorphism have been used in a study oflinkage between the ^(G) γ-globin and parathyroid loci (Cui et al.,1989, Proc. Natl. Acad. Sci. 6:9389-9393). In both of these studies, theobserved HindIII site polymorphism corresponds to the present A and Balleles.

Identification of individuals is possible with genetic typing. The useof polymerase chain reaction (PCR) amplification and nucleotide probesto detect even single nucleotide changes in a gene sequence hasrevolutionized the field of forensic serology (see Reynolds andSensabaugh 1991, Anal. Chem. 63:2-15). With PCR and other nucleic acidamplification methods, DNA typing can now be done with samples thatcontain insufficient DNA for typing by any other means; single hairs,for example, provide enough DNA for PCR-based DNA typing. (Higuchi etal., 1988, Nature 332:543-546).

SUMMARY OF THE INVENTION

The present invention provides methods and reagents for determining anindividual's genotype at the ^(G) γ-globin locus with respect to a setof three alleles. Only two alleles, defined by a HindIII restrictionenzyme cleavage site polymorphism, have been described in the prior art.The discovery of a third allele and the creation of sequence-specific,oligonucleotide probes complementary to the three alleles form the basisof the present invention, which provides a relatively rapid, convenient,and accurate genotyping method. In this tri-allelic system, allele Acorresponds to the allele containing the HindIII site (+HindIII),whereas alleles B and C subdivide the class of alleles lacking theHindIII site (-HindIII) into two distinct alleles. The specificsequences defining the A, B, and C alleles are provided below.

One aspect of the invention relates to a process for determining anindividual's genotype at the ^(G) γ-globin locus from a samplecontaining nucleic acid obtained from the individual. The processcomprises hybridizing the sample nucleic acid with a panel ofsequence-specific oligonucleotide (SSO) probes; each of the probes iscomplementary to a variant segment of the ^(G) γ-globin locus definingthe A, B, or C allele. The hybridization is carried out under conditionssuch that the SSO probes bind to the nucleic acid to form stable hybridduplexes only if the hybridizing region of each of the probes is exactlycomplementary to the nucleic acid. The hybrids formed between thenucleic acid and the SSO probes can then be detected. The sample cancontain amplified nucleic acids, so long as the relevant regions ofnucleic acid have been amplified; any of the known methods forincreasing the copy number of a region of nucleic acid in vitro can beused to amplify the nucleic acid.

Another aspect of the invention relates to SSO probes useful fordiscriminating between the three alleles that may be present in thesample.

A third aspect of the invention relates to kits useful for determiningthe ^(G) γ-globin genotype of an individual. These kits take a varietyof forms and comprise one or more SSO probes and, in one embodiment,comprise a panel of probes sufficient to determine the ^(G) γ-globingenotype relative to the A, B, and C alleles and instructions fordetermining the genotype by using the kit ingredients. The kits can alsocomprise one or more amplification reagents, e.g., primers, polymerase,buffers, and nucleoside triphosphates.

A fourth aspect of the invention relates to forensic methods todetermine the probable orgin of a biological sample. The presentthree-allele system substantially improves the discriminative power of^(G) γ-globin DNA typing methodology and will have an important impacton forensic methodology.

To aid in understanding the invention, several terms are defined below.

The terms "^(G) γ-globin gene" and "^(G) γ-globin locus" refer to atranscribed region of DNA that contains the coding sequence for the ^(G)γ-globin protein and the untranslated intervening sequences.

The term "alleles" refers to variants of the nucleotide sequence of agene. An allele is defined by the presence of a specific subsequence. Anallele may consist of a set of sequence variants, all of which containthe specific subsequence that defines the allele.

The term "allele A" refers to sequence variants of the ^(G) γ-globingene that contain the HindIII restriction enzyme cleavage site sequence5'-AAGCTT.

The term "allele B" refers to sequence variants of the ^(G) γ-globingene that contain the sequence 5'-AAGCTG in place of the HindIIIrestriction site of allele A.

The term "allele C" refers to sequence variants of the ^(G) γ-globingene that contain the sequence 5'-AATCTT in place of the HindIIIrestriction site of allele A.

The term "genotype" refers to a description of the alleles of a genecontained in an individual or a sample.

The term "oligonucleotide" refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, such as primers, probes,nucleic acid fragments to be detected, and nucleic acid controls. Theexact size of an oligonucleotide depends on many factors and theultimate function or use of the oligonucleotide. Oligonucleotides can beprepared by any suitable method, including, for example, cloning andrestriction of appropriate sequences and direct chemical synthesis by amethod such as the phosphotriester method of Narang et al., 1979, Meth.Enzymol, 68:90-99; the phosphodiester method of Brown et al., 1979,Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucageet al., 1981, Tetrahedron Lett. 22:1859-1862; and the solid supportmethod of U.S. Pat. No. 4,458,066.

The terms "polymorphic" and "polymorphism" refer to the condition inwhich two or more variants of a specific DNA sequence can be found in apopulation.

The terms "polymorphic gene" and "polymorphic region" refer to thatregion of the DNA where a polymorphism occurs.

The term "primer" refers to an oligonucleotide, whether natural orsynthetic, capable of acting as a point of initiation of DNA synthesisunder conditions in which synthesis of a primer extension productcomplementary to a nucleic acid strand is induced, i.e., in the presenceof four different nucleoside triphosphates and an agent forpolymerization (i.e., DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature. A primer is preferablya single-stranded oligodeoxyribonucleotide. The appropriate length of aprimer depends on the intended use of the primer but typically rangesfrom 15 to 25 nucleotides. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with atemplate.

The term "primer" may refer to more than one primer, particularly in thecase where there is some ambiguity in the information regarding one orboth ends of the target region to be amplified. For instance, if anucleic acid sequence is inferred from a protein sequence, a "primer" isactually a collection of primer oligonucleotides containing sequencesrepresenting all possible codon variations based on the degeneracy ofthe genetic code. One of the primers in this collection will behomologous with the end of the target sequence. Likewise, if a"conserved" region shows significant levels of polymorphism in apopulation, mixtures of primers can be prepared that will amplifyadjacent sequences. A primer can be labeled, if desired, byincorporating a label detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³² P, fluorescent dyes, electron-dense reagents, enzymes(as commonly used in ELISAS), biotin, or haptens and proteins for whichantisera or monoclonal antibodies are available. A label can also beused to "capture" the primer, so as to facilitate the immobilization ofeither the primer or a primer extension product, such as amplified DNA,on a solid support.

The terms "restriction endonucleases" and "restriction enzymes" refer toenzymes, usually of bacterial origin, that cut double-stranded DNA at ornear a specific nucleotide sequence.

The terms "sequence-specific oligonucleotide" and "SSO" refer tooligonucleotides that have a sequence, called a "hybridizing region,"exactly complementary to the sequence to be detected, typicallysequences characteristic of a particular allele, which under"sequence-specific, stringent hybridization conditions" will hybridizeonly to that exact complementary target sequence. Depending on thesequences being analyzed, one or more sequence-specific oligonucleotidesmay be employed for each sequence. The terms "probe" and "SSO probe" areused interchangeably with SSO.

The term "target region" refers to a region of a nucleic acid which isto be analyzed and usually includes a polymorphic region.

The term "thermostable polymerase enzyme" refers to an enzyme that isrelatively stable to heat and catalyzes the polymerization of nucleosidetriphosphates to form primer extension products that are complementaryto one of the nucleic acid strands of the target sequence. The enzymeinitiates synthesis at the 3'-end of the primer and proceeds in thedirection toward the 5' end of the template until synthesis terminates.A purified thermostable polymerase enzyme is described more fully inU.S. Pat. No. 4,889,818, incorporated herein by reference, and iscommercially available from Perkin-Elmer Cetus Instruments (PECI,Norwalk, CT).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides processes and reagents for determiningthe ^(G) γ-globin genotype of an individual. In part, the inventionresults from the discovery of an additional polymorphism in the regionspecifying the HindIII site. The additional polymorphism was firstobserved after screening polymerase chain reaction (PCR) amplified DNAcontaining the polymorphic region of the ^(G) γ-globin for the presenceor absence of the HindIII site with sequence-specific oligonucleotideprobes. A significant fraction of the samples from Black individuals didnot hybridize to either probe, indicating that these individuals werehomozygous for an unknown allele. The existence of this third allele wasconfirmed by direct sequencing of the PCR products from the aboveamplification. The defining sequences for these three alleles, heredesignated alleles A, B, and C, are provided above. The SSO probesprovided by the invention enable one to detect the variant genotypes byinference from the pattern of binding of a panel of probes. In apreferred embodiment, each probe of the panel is specific for adifferent allele of the ^(G) γ-globin gene. Table VI providesdesignations of illustrative probes of the invention; the correspondingsequences are provided in the sequence listing section.

As described above, the A, B, and C alleles of the ^(G) γ-globin genecan be distinguished from one another by the polymorphic sequencecorresponding to the HindIII site of the A allele. In an alternativeembodiment of the invention, however, one can distinguish certain Calleles from certain A and B alleles by detecting a polymorphismadjacent to the HindIII polymorphism. In all reported sequences for Aand B alleles and in all C alleles sequenced by the present inventors,the HindIII polymorphism is contained within the larger sequences shownin Table I below:

                  TABLE I                                                         ______________________________________                                        Allele  Sequence                                                              ______________________________________                                        A       5'-AAGCTTGGTGTGTAG-3' (SEQ ID NO: 56)                                 B       5'-AAGCTGGGTGTGTAG-3' (SEQ ID NO: 57)                                 C       5'-AATCTTGGTGTGTAA-3' (SEQ ID NO: 58)                                 ______________________________________                                    

Thus, one can distinguish the C allele shown above from the A and Balleles shown above by use of SSO probes specific for the A:Gpolymorphism at the 3' end of the sequence. Such probes would typicallycontain sequences that, in addition to hybridizing to the A:Gpolymorphic base, would hybridize to sequences 3' to the polymorphicbase. Such 3' sequences are not shown in the Table above but can beobtained from Shiokawa et al., supra. There may be C alleles, however,that do not contain this single base variation, but could still bedistinguished by SSO probes specific for the HindIII polymorphism.

In a preferred embodiment of the invention, the process for DNA basedtyping of ^(G) γ-globin genotype comprises amplifying a nucleic acidsequence which contains the variable portion of a ^(G) γ-globin gene,determining the variant ^(G) γ-globin sequence present with SSO probes;and inferring the ^(G) γ-globin genotype from the pattern of binding ofthe SSO probes to the amplified target sequence. To facilitate practiceof this preferred embodiment, the present invention provides primers foramplifying by a PCR the ^(G) γ-globin target region. The designations ofthese primers are listed in Table VII; the sequences of these primersare in the sequence listing section.

In this preferred method, a sample containing nucleic acid is obtainedfrom an individual whose ^(G) γ-globin genotype is to be determined. Anytype of tissue containing ^(G) γ-globin nucleic acid may be used forpurposes of the present invention. Because the genotyping methods of thepresent invention can utilize amplified nucleic acids, and because thePCR technique can amplify extremely small quantities of nucleic acid,even samples containing only a few copies of the ^(G) γ-globin gene canbe typed for the ^(G) γ-globin variants by the present method. Forinstance, even a single hair contains enough DNA for purposes of thepresent invention, as evidenced by the DQα DNA typing methods describedby Higuchi et al., supra.

In general, the nucleic acid in the sample will be DNA, most usuallygenomic DNA. However, the present invention can also be practiced withother nucleic acids, such as messenger RNA or cloned DNA, and thenucleic acid may be either single-stranded or double-stranded in thesample and still be suitable for purposes of the present invention.Those skilled in the art recognize that whatever the nature of thenucleic acid, the nucleic acid can be typed by the present method merelyby taking appropriate steps at the relevant stage of the process. If PCRis used to amplify the nucleic acid in the sample, then the sample willusually comprise double-stranded DNA after amplification and beforeprobe hybridization.

As noted above, in a preferred embodiment, the ^(G) γ-globin typingmethod and probes of the invention are used in conjunction withPCR-amplified target DNA. Those practicing the present invention shouldnote, however, that amplification of ^(G) γ-globin target sequences in asample may be accomplished by any known method, such as ligase chainreaction (LCR), transcription amplification and self-sustained sequencereplication, each of which provides sufficient amplification so that thetarget sequence may be detected by nucleic acid hybridization to an SSOprobe. Alternatively, methods which amplify the probe to detectablelevels can be used, such as Qβ-replicase amplification. The term "probe"encompasses the sequence-specific oligonucleotides used in the aboveprocedures; for instance, the two or more oligonucleotides used in LCRare "probes" for purposes of the present invention, even though someembodiments of LCR only require ligation of the probes to indicate thepresence of an allele.

Although the PCR process is well known in the art (see U.S. Pat. Nos.4,683,195; 4,683,202; and 4,965,188, each of which is incorporatedherein by reference) and although commercial vendors, such as PECI, sellPCR reagents and publish PCR protocols, some general PCR information isprovided below for purposes of clarity and full understanding of theinvention to those unfamiliar with the PCR process.

To amplify a target nucleic acid sequence in a sample by PCR, thesequence must be accessible to the components of the amplificationsystem. In general, this accessibility is ensured by isolating thenucleic acids from the sample. A variety of techniques for extractingnucleic acids from biological samples are known in the art. For example,see those described in Higuchi et al., 1989 in PCR Technology (Erliched., Stockton Press, New York). Alternatively, if the sample is fairlyreadily disruptable, the nucleic acid need not be purified prior toamplification by the PCR technique, i.e., if the sample is comprised ofcells, particularly peripheral blood lymphocytes or amniocytes, lysisand dispersion of the intracellular components may be accomplishedmerely by suspending the cells in hypotonic buffer.

Because the nucleic acid in the sample is first denatured (assuming thesample nucleic acid is double-stranded) to begin the PCR process, andbecause simply heating some samples results in the disruption of cells,isolation of nucleic acid from the sample can sometimes be accomplishedin conjunction with strand separation. Strand separation can beaccomplished by any suitable denaturing method, however, includingphysical, chemical, or enzymatic means. Typical heat denaturationinvolves temperatures ranging from about 80° C. to 105° C. for timesranging from seconds to about 1 to 10 minutes. Strand separation mayalso be induced by a helicase, an enzyme capable of exhibiting helicaseactivity. For example, the enzyme RecA has helicase activity in thepresence of ATP. The reaction conditions suitable for strand separationby helicases are known in the art (see Kuhn Hoffman-Berling 1978,CSH-Quantitative Biology 43:63-67; and Radding 1982, Ann. Rev. Genetics16:405-436).

As noted above strand separation may be accomplished in conjunction withthe isolation of the sample nucleic acid or as a separate step. In thepreferred embodiment of the PCR process, strand separation is achievedby heating the reaction to a sufficiently high temperature for aneffective time to cause the denaturation of the duplex, but not to causean irreversible denaturation of the polymerase (see U.S. Pat. No.4,965,188). No matter how strand separation is achieved, however, oncethe strands are separated, the next step in PCR involves hybridizing theseparated strands with primers that flank the target sequence. Theprimers are then extended to form complementary copies of the targetstrands, and the cycle of denaturation, hybridization, and extension isrepeated as many times as necessary to obtain the desired amount ofamplified nucleic acid.

As noted above, the present invention provides PCR primers for theamplification of a target region of the ^(G) γ-globin gene. Theseprimers are complementary to sequences in conserved regions that flankthe polymorphic region wherein the ^(G) γ-globin sequence variations arelocated. For successful PCR amplification, the present primers aredesigned so that the position at which each primer hybridizes along aduplex sequence is such that an extension product synthesized from oneprimer, when separated from the template (complement), serves as atemplate for the extension of the other primer to yield an amplifiedsegment of nucleic acid of defined length. Moreover, primers areprovided that will bind preferentially to the ^(G) γ-globin region underselective annealing conditions.

Template-dependent extension of primers in PCR is catalyzed by apolymerizing agent in the presence of adequate amounts of fourdeoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP) in areaction medium comprised of the appropriate salts, metal cations, andpH buffering system. Suitable polymerizing agents are enzymes known tocatalyze template-dependent DNA synthesis. For example, if the templateis RNA, a suitable polymerizing agent to convert the RNA into acomplementary, copy-DNA (cDNA) sequence is reverse transcriptase (RT),such as avian myeloblastosis virus RT and Thermus thermophilus DNApolymerase, a thermostable DNA polymerase with reverse transcriptaseactivity marketed by PECI. Once the target for amplification is DNA,suitable polymerases include, for example, E. coli DNA polymerase I orits Klenow fragment, T₄ DNA polymerase, and Taq polymerase, a heatstable DNA polymerase isolated from Thermus aquaticus and commerciallyavailable from PECI. The latter enzyme is widely used in theamplification and sequencing of nucleic acids. The reaction conditionsfor using Taq polymerases are known in the art and are described inGelfand, 1989, in PCR Technology, supra.

The PCR method can be performed in a step-wise fashion, where after eachstep new reagents are added, or in a fashion where all of the reagentsare added simultaneously, or in a partial step-wise fashion, where freshor different reagents are added after a given number of steps. Forexample, if strand separation is induced by heat, and the polymerase isheat-sensitive, then the polymerase has to be added after every round ofstrand separation. However, if, for example, a helicase is used fordenaturation, or if a thermostable polymerase is used for extension,then all of the reagents may be added initially, or, alternatively, ifmolar ratios of reagents are of consequence to the reaction, thereagents may be replenished periodically as they are depleted by thesynthetic reaction.

Those skilled in the art will know that the PCR process is most usuallycarried out as an automated process with a thermostable enzyme. In thisprocess, the temperature of the reaction mixture is cycled thorugh adenaturing region, a primer annealing region, and a reaction region. Amachine specifically adapted for use with a thermostable enzyme iscommercially available from PECI.

Those skilled in the art will also be aware of the problem ofcontamination of a PCR by the amplified nucleic acid from previousreactions. Methods to reduce this problem are provided in U.S. patentapplication Ser. No. 609,157, incorporated herein by reference.

Amplification of the DNA sequences of the alleles of the ^(G) γ-globingene is a useful, but not a necessary, step in determining the ^(G)γ-globin genotype of an individual. Specific probe hybridization,however, is an important step in successful performance of the presentmethods. The sequence-specific, oligonucleotide probes of the presentinvention are designed to hybridize specifically with a particularvariant segment of a ^(G) γ-globin allele and to have destabilizingmismatches with the other variant sequences known for the particularsegment and work equally well with amplified or unamplified genomic DNA.Under stringent hybridization conditions, the probes hybridizespecifically only to exactly complementary sequences in the variantsegment of the ^(G) γ-globin alleles. These SSO probes allow forsequence specific hybridization and comprise a hybridizing region thatis preferably in the range of 10 to 30 bases, more preferably 12 to 24bases, in length.

The assay methods for detecting hybrids formed between SSO probes andnucleic acid sequences can require that the probes contain additionalfeatures in addition to the hybridizing region. For example, if theprobe is first immobilized, as in the "reverse" dot blot formatdescribed below, the probe can also contain long stretches of poly-dTthat can be fixed to a nylon support by irradiation, a techniquedescribed in more detail in PCT Patent Publication No. 89/11548,incorporated herein by reference.

The probes of the invention can be synthesized and labeled using thetechniques described above for synthesizing oligonucleotides. Forexample, the probe may be labeled at the 5'-end with ³² P by incubatingthe probe with ³² P-ATP and kinase. A suitable non-radioactive label forSSO probes is horseradish peroxidase (HRP). Methods for preparing anddetecting probes containing this label are described in the Examplesbelow and in U.S. Pat. Nos. 4,914,210, and 4,962,029; the latter patentsare incorporated herein by reference. For additional information on theuse of such labeled probes, see U.S. Pat. No. 4,789,630; Saiki et al.,1988, N. Eng. J. Med. 319:537-541; and Bugawan et al., 1988, BioTechnology 6:943-947, each of which is incorporated herein by reference.Useful chromogens include red leuco dye and3,3',5,5'-tetramethylbenzidine (TMB).

The probes of the invention can be used to identify the allelicsequences present in a sample by determining the SSO probes that bind tothe ^(G) γ-globin sequences present in the sample. Suitable assaymethods for purposes of the present invention to detect hybrids formedbetween SSO probes and nucleic acid sequences in a sample are known inthe art. For example, the detection can be accomplished using a dot blotformat, as described in the Examples. In the dot blot format, theunlabeled amplified sample is bound to a solid support, such as amembrane, the membrane incubated with labeled probe under suitablehybridization conditions, the unhybridized probe removed by washing, andthe filter monitored for the presence of bound probe. When multiplesamples are analyzed with few probes, the dot blot format is quiteuseful.

An alternate method that is quite useful when large numbers of differentprobes are to be used is a "reverse" dot blot format, in which theamplified sequence contains a label, and the probe is bound to the solidsupport. In this format, the unlabeled SSO probes are bound to themembrane and exposed to the labeled sample under appropriately stringenthybridization conditions. Unhybridized labeled sample is then removed bywashing under suitably stringent conditions, and the filter is thenmonitored for the presence of bound sequences.

Another suitable assay system is described in U.S. patent applicationSer. No. 563,758, incorporated herein by reference, in which a labeledprobe is added during the PCR amplification process. Any SSO probe whichhybridizes to target DNA during each synthesis step is degraded by the5' to 3' exonuclease activity of e.g., Taq polymerase. The degradationproduct from the probe is then detected. Thus, the presence of thebreakdown product indicates that the hybridization between the SSO probeand the target DNA occurred.

Whatever the method for determining which SSO probes of the inventionhybridize to ^(G) γ-globin sequences in a sample, the central feature ofthe typing method involves the identification of the ^(G) γ-globinalleles present in the sample by analyzing the pattern of binding of apanel of SSO probes. The specific application will determine whichprobes are used in a panel. For instance, if only the presence orabsence of the C allele is of interest, a single probe specific for theC allele is adequate. The three possible genotypes of haploid cells(sperm cells, for example) can be determined using only two probes, onespecific for each of two of the alleles. The third allele is thenrecognized by the absence of probe hybridization. In diploid cells,however, heterozygotes involving this third allele cannot be detectedwith only probes for the other two alleles. The feasibility of usingsingle sperm for DNA typing is demonstrated in Li et al, 1988, Nature335:441-417.

Another typing method useful for hapoid cells using only two probes isusing probes that hybridize to only two out of the three alleles. Thisis achieved by using probes that are complementary to only part of theregion where the HindIII site is located, as exemplified in Table IIbelow. Only the ends of the hydridization regions of the probesextending into the HindIII site are shown; the rest of the probe isindicated by arrows. Probe 1, which is complementary to all of theHindIII site except for the last position where the T to G substitutiondefining the B allele occurs, will not bind to the C allele, but willbind to both the A and B allele. Similarly, probe 2 will hybridize tothe A and C alleles, but not the B allele. Together, these probes aresufficient to determine the genotype of a haploid cell; both probeshybridizing indicates allele A is present, only probe 1 indicates alleleB, and only probe 2 indicates allele C.

                  TABLE II                                                        ______________________________________                                         ##STR1##                                                                     ______________________________________                                    

DNA typing of ^(G) γ-globin alleles is useful for many differentpurposes. For example, DNA typing methods now play a significant role inthe important area of individual identification, whether for solvingcrimes, as when the identify of a criminal or victim is established bylinking an individual with evidence left at the scene of a crime, or forsolving other issues of a non-criminal nature, as when biologicalmaterial is used to determine the maternity or paternity of anindividual.

The DNA sequences provided above are an important aspect of the presentinvention. Although only one strand of the sequence is shown, those ofskill in the art recognize that the other strand of the sequence can beinferred from the information depicted above. This information enablesthe construction of the probes and primers of the invention.Illustrative probes and primers of the invention are shown in thesequence listing. Tables VI and VII gives the corresponding designationsfor each sequence.

The present invention also relates to kits, multicontainer unitscomprising useful components for practicing the present method. A usefulkit can contain SSO probes for the ^(G) γ-globin gene. In some cases,the SSO probes may be fixed to an appropriate support membrane. The kitcan also contain primers for PCR, as such primers are useful in thepreferred embodiment of the invention. These primers will amplify apolymorphic region of the ^(G) γ-globin gene. Other optional componentsof the kit include, for example, an agent to catalyze the synthesis ofprimer extension products, the substrate nucleoside triphosphates, meansused to label (for example, an avidinenzyme conjugate and enzymesubstrate and chromogen if the label is biotin), and the appropriatebuffers for PCR or hybridization reactions. In addition to the abovecomponents, the kit can also contain instructions for carrying out thepresent method.

The examples of the present invention presented below are provided onlyfor illustrative purposes and not to limit the scope of the invention.Numerous embodiments of the invention within the scope of the claimsthat follow the examples will be apparent to those of ordinary skill inthe art from reading the foregoing text and following examples.

EXAMPLE 1 ^(G) γ-globin Typing-Dot Blot Format

For typing samples with the panel of probes, 0.1 μg of human genomic DNAis amplified using reaction constituents as described in Saiki et al.,1988, Science 239:487-490, incorporated herein by reference. Theprimers, RS287 (SEQ ID NO:32) and RS288 (SEQ ID NO:33), are present inthe reaction mixture at 0.25 μM. These primers produce a 345 base-pair(bp) fragment. Samples are amplified for 32 cycles using the reactionconditions described above. All samples are overlaid with 100 μl of highgrade mineral oil (Sigma, St. Louis, MO) to prevent evaporation. One canalso use the hot start methodology described in U.S. patent applicationSer. No. 481,501, filed Feb. 16, 1990, and incorporated herein byreference, in performing the PCR amplification.

Before the first cycle, the sample is incubated at 72° C. for 30seconds. The thermal profile for each of the 32 cycles comprisesincubations at the following temperatures for the indicated times: 60seconds at 94° C. (to denature the DNA strands), 30 seconds at 60° C.(to anneal the primers), and 30 seconds at 72° C. (to extend theprimers). The PECI Thermal Cycler is programmed to incubate the samplesat 72° C. for 10 minutes after the last cycle to ensure that the finalextension is complete.

After amplification, a small portion of the amplified DNA is denaturedand applied to a series of nylon filters; each filter is then hybridizedto one of the labelled probes. Each of the SSO probes SN27 (SEQ IDNO:1), SN26 (SEQ ID NO:18), and RS339 (SEQ ID NO:23) is covalentlyconjugated to horseradish peroxidase (HRP) and provides a means ofnonisotopic detection in the presence of a chromogenic orchemiluminescent substrate.

Thus, 5 μl of each amplified DNA sample are mixed with 100 μl of amixture composed of 0.4M NaOH and 25 mM EDTA, and the resulting mixtureis applied to BioDyne B nylon filters (Pall Corp., Glen Cove, NY) usinga dot-blot manifold (Bio Rad, Richmond, CA). The filters are rinsed witha mixture of 10 mM Tris-HCl and 0.1 mM EDTA, at pH 8.0, and dried onWhatman 3MM paper. The DNA is immobilized on the nylon filter byultraviolet irradiation at a flux of 55 mJ/cm² with a Stratalinker™(Stratagene, La Jolla, CA) UV light box.

All filters are hybridized in 2× SSPE (saline sodium phosphate EDTA), 5×Denhardt's solution, and 0.5% SDS with 2 pmoles of HRP-SSO probe per 5ml of hybridization solution for 15 min. at 42° C. Horseradishperoxidase conjugated oligonucleotides are prepared as described byLevenson and Chang, 1989, in PCR Protocols: A Guide to Methods andApplications, Innis et al., eds., Academic Press. San Diego):92-112,incorporated herein by reference, and Saiki et al., 1988, N. Eng. J.Med. 319:537-541. Filters for each probe are washed in 10 ml of the SSPEsolution at 55° C.

After washing, filters to be developed with a chromogenic dye substrateare rinsed in 100 mM sodium citrate, pH 5.0, then placed in 100 mMsodium citrate, pH 5.0, containing 0.1 mg/ml of3,3',5,5'-tetramethylbenzidine per milliliter (Fluka) and 0.0015 percenthydrogen peroxide, and incubated with gentle agitation for 10 to 30minutes at room temperature. Developed filters are rinsed in water andimmediately photographed. The TMB detection system is prepared and usedsubstantially as described in AmpliType® DQalpha DNA typing kit marketedby PECI. In another embodiment, filters are developed with thechemiluminescent detection system (ECL; Amersham, Arlington Heights,IL). Filters are rinsed in PBS for 5 minutes and placed in the ECLsolution for 1 minute with gentle agitation. Filters are then exposed toX-ray film at room temperature for 1 to 5 minutes.

EXAMPLE 2 ^(G) γ-globin Typing--Reverse Dot Blot Format

In this embodiment of the invention, the ^(G) γ-globin probes are fixedto a membrane, and the amplified target DNA is hybridized to themembrane-bound probe as described in Saiki et al., 1989, Proc. Natl.Acad. Sci. 86:6230-6234 and in the AmpliType® DQalpha DNA typing kitmarketed by PECI. The set of typing probes is designed so that eachprobe will hybridize to a specific target sequence at the sametemperature and salt concentration (and stay hybridized under the samewash conditions) as all other probes in the set. The PCR primers used inthe amplification are biotinylated, as described in Levenson and Chang,1989, supra, so that any amplified DNA that hybridizes to themembrane-bound probes can be easily detected.

In one embodiment, detection is carried out by reacting streptavidinconjugated horseradish peroxidase (SA-HRP) with any biotinylated,amplified DNA hybridized to the membrane-bound probe. The HRP thusbecomes bound, through the SA-biotin interaction, to the amplified DNAand can be used to generate a signal by a variety of well known means,such as the generation of a colored compound, e.g., by the oxidation oftetramethylbenzidine (see U.S. Pat. No. 4,789,630).

Although the probes can be fixed to the membrane by any means, apreferred method involves "tailing" an oligonucleotide probe about 13 to25 nucleotides in length (the hybridizing region) with a much longersequence of poly-dT. The resulting poly-dT "tail" can then be reactedwith amine groups on a nylon membrane to fix the probe covalently to themembrane. This reaction can be facilitated by UV irradiation.

Terminal deoxyribonucleotidyl transferase (TdT, Ratliff Biochemicals;for the reactions below assume a concentration of about 120 Units/μl,which is 100 pmole/μl) can be used to create a poly-dT tail on a probe,although one can also synthesize the tailed probe on a commerciallyavailable DNA synthesizer. When one uses a DNA synthesizer to make thetailed probe, however, one should place the tail on the 5' end of theprobe, so that undesired premature chain termination occurs primarily inthe tail region.

TdT reactions should be carried out in a volume of about 100 μlcontaining 1X TdT salts, 200 pmole of oligonucleotide, 800 μM dTT, and60 units of TdT. 10X TdT salts is 1,000 mM K-cacodylate, 10 mM CoCl₂, 2mM dithiothreitol, 250 mM Tris-Cl, pH 7.6, and is prepared as describedby Roychoudhury an Wu, Meth. Enzymol. 65: 43-62, incorporated herein byreference. A 10× stock solution of 8 mM dTTP can be prepared(neutralized to pH 7 with NaOH) for convenience.

The TdT reaction should be carried out at 37° C. for two hours and thenstopped by the addition of 100 μl of 10 mM EDTA, pH 8. The finalconcentration of tailed oligonucleotide is 1 μM (1 pmole/μl), and thelength of the homopolymer tail is about 400 residues. Tail length can bechanged by adjusting the molar ratio of dTTP to oligonucleotide. Thetailed probes can be stored at -20° C. until use.

Two types of nylon membrane are preferred for the reverse dot blotformat: Biodyne™ nylon membrane, 0.45 micron pore size, manufactured byPall; and Biotrans™ nylon membrane, 0.45 micron pore size, manufacturedby ICN. The probes can be spotted onto the membrane very convenientlywith the Bio-Dot™ dot blot apparatus manufactured by BioRad. Each probeis spotted onto a unique, discrete location onto the membrane. About 2to 10 picomoles of each tailed probe is premixed with 50-100 μl of TEbuffer before application to the dot blot apparatus. After dot blotting,the membrane is briefly placed on absorbent paper to draw off excessliquid.

The membrane is then placed inside a UV light box, such as theStratalinker™ light box manufactured by Stratagene and exposed to 50 to60 millijoules/cm² of flux at 254 nm to fix the tailed probe to thenylon membrane. After a brief rinse (for about 15 minutes inhybridization solution) to remove unbound probe, the membrane is thenready for hybridization with biotinylated PCR product. One-half to onepicomole (one-quarter to one-half of a typical, 100 μl PCR mixture) ofPCR product is added to each probe panel for hybridization. About 50 μlof SA-HRP, commercially available from PECI, can be added at this timefor convenience, but better signals will result if a separate SA-HRPincubation and wash, at room temperature, is performed after thehybridization step but before the stringency wash.

Hybridization is typically carried out at 55° C. for 30 minutes in awater bath and with hybridization buffer composed of 0.5% SDS and 5×SSPE. Biotin binding is carried out in the hybridization buffer for 10minutes at 55° C. Stringency washing is carried out at 55° C. for 10minutes in a water bath and with wash solution composed of 0.1% SDS and2.5× SSPE. A post-wash of 1× PBS at room temperature for 30 minutes canenhance signal quality.

The hybridization region of the biotinylated primers for the reverse dotblot method are the same as the primers of Example 1. Note that one orboth of the primers can be biotinylated in an amplification and that theprimers can be used for amplification with any detection format.

The hybridizing regions of the tailed probes and biotinylated primersfor use in the reverse dot blot method are the same as the probes andprimers of Example 1.

EXAMPLE 3 FREQUENCIES OF THE ^(G) γ-GLOBIN ALLELES

Samples from three different populations were typed at the ^(G) γ-globinlocus: 20 from a Caucasian population, fourteen from a Hispanicpopulation, and 34 from a Black population. The typing at the ^(G)γ-globin locus was done simultaneously with typing at LDL, D7S8, and Gcloci. In addition, primers for amplifying a region of the DQ locus wereadded during the amplification step. The results of the ^(G) γ-globinDNA typing are shown below.

Amplification was done substantially as in Example 1 with themodifications described below. The amplification reagents "premix"solution used comprised the following: 200 μl of 10X Taq buffer; 200 μlof dNTP's (8 mM solution); 50 μl each of primers (10 μM solution) RS287(SEQ ID NO:32), RS288 (SEQ ID NO:33), SN58, SN59, RS175, RS176, RS227,RS228, RS134, and RS135; 1,084 μl of H₂ O; and 16 μl of Taq polymerase(5 units/μl solution). The primers other than RS287 and RS288 were foramplification of regions of the other loci. About 100 ng of genomic DNAwere added to 100 μl of premix solution and amplified using thetemperature profile of Example 1.

The probes used for typing at the ^(G) γ-globin locus were SN27 (SEQ IDNO:1), SN26 (SEQ ID NO:18), RS339 (SEQ ID NO:23) for the A, B, and Calleles, respectively. The probes designated RS179, RS180, RS229, RS230,RS318, RS319, and RS320 were used for typing at the other three loci.The reverse dot-blot detection scheme was used substantially asdescribed in Example 2, except that hybridization was carried out for 15minutes at 55° C. and the subsequent stringency wash was for 10 minutesat 55° C. Filters were developed substantially as in Example 1, but with2 mg/ml TMB and 3% H₂ O₂.

Allele and genotype frequency estimates for the three populations aregiven in Table II below. Numbers may not add to 1 due to round-offerrors. The frequency of the C allele in the Black population issubstantial. The usefulness of the ^(G) γ-globin locus for individualidentification is greatly enhanced by the discovery of a third allelepresent in the Black population at relatively high frequencies.

                  TABLE III                                                       ______________________________________                                        Popu-                                                                         lation A     B      C    AA   AB   AC   BB   BC   CC                          ______________________________________                                        Cau-   .60   .375   .025 .40  .15  0    .4   .05  0                           casion                                                                        Hispanic                                                                             .36   .54    .10  .14  .29  .14  .36  .07  0                           Black  .38   .29    .32  .12  .26  .26  .09  .18  .12                         ______________________________________                                    

EXAMPLE 4 Polymarker Kit

In this embodiment, the ^(G) γ-globin typing system is used in thePolymarker™ kit produced by Cetus Corporation as one part of a six genetyping system with particular application in the field of forensics. TheDNA type of a suspected individual is compared to the type determinedfrom the sample of unknown origin to determine if the sample could haveoriginated from the suspect. The increased power of discrimination thatcomes from typing multiple loci and as many alleles at each loci asconveniently possible increases the effectiveness of the test in theforensic setting.

The protocol is substantially as in Examples 2 and 3, above, with somedifferences noted as follows. The concentrations in the reaction mixtureare 0.25 μM for each of the primers, 0.2 mM for each of the dNTPs, and 4units per reaction of Taq polymerase. The 32 cycles of amplification(94° C., 1 minute; 60° C., 30 seconds; 72° C., 30 seconds) are followedby a final 7 minute extension at 72° C.

In this embodiment, six loci are simultaneously typed. The designationand sequence identification numbers of the primers and probes for allthe loci used are listed below. The probes are fixed to a Nylon membrane(Pall Biodyne B™).

                  TABLE IV                                                        ______________________________________                                        Primer Pairs                                                                  Gene   Primer 1         Primer 2                                              ______________________________________                                        Gγ-                                                                            RS287 (SEQ ID NO: 32)                                                                          RS412 (SEQ ID NO: 33)                                 globin                                                                        LDLr   RS175 (SEQ ID NO: 36)                                                                          RS176 (SEQ ID NO: 37)                                 DQα                                                                            RS134 (SEQ ID NO: 38)                                                                          RS135 (SEQ ID NO: 39)                                 Glyco- RS362 (SEQ ID NO: 40)                                                                          RS364 (SEQ ID NO: 41)                                 phorin A                                                                      D7S8   RS227 (SEQ ID NO: 42)                                                                          RS383 (SEQ ID NO: 43)                                 Gc     SN58 (SEQ ID NO: 44)                                                                           SN59 (SEQ ID NO: 45)                                  ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        DNA Probe Strips                                                                                           Seq ID No. of                                    Gene       Allele   T-Tail   Hybridization Region                             ______________________________________                                        Gγ-globin                                                                          A        200T      1                                                          B        100T     22                                                          C        100T     30                                               LDLr       A        200T     46                                                          B        100T     47                                               Glycophorin (M)                                                                          A        100T     48                                               A (N)      B        100T     49                                               D7S8       A        100T     50                                                          B        100T     51                                               Gc (2)     A        100T     52                                               (1F)       B        100T     53                                               (1S)       C        100T     54                                               DQα All Probe                                                                      S        200T     55                                               ______________________________________                                    

EXAMPLE 5 Primers and Probes

Those of skill in the art recognize that primers and probes useful inthe present methods can vary somewhat in length and therefore insequence. Tables VI and VII below show a variety of illustrative primersand probes useful in the present method.

                                      TABLE VI                                    __________________________________________________________________________    Probes                                                                        __________________________________________________________________________     ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                     __________________________________________________________________________

                  TABLE VII                                                       ______________________________________                                        Primers      Seq. ID No.                                                      ______________________________________                                        RS173        31                                                               RS287        32                                                               RS288        33                                                               RS410        34                                                               RS412        35                                                               ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 58                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: Other nucleic acid                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CACACCAAGCTTCCAC16                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: Other nucleic acid                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CACACCAAGCTTCCACC17                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TACACACCAAGCTTCCA17                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                              (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AACACACCAAGCTTCCAC18                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                              (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CACCAAGCTTCCACCC16                                                            (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                              (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      ATAACTACACACCAAGC17                                                           (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      ACACCAAGCTTCCACC16                                                            (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single stranded                                            (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      CAAGCTTCCACCCAGA16                                                            (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single stranded                                            (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      CACCAAGCTTCCACCT16                                                            (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single stranded                                            (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     CACACCAAGCTTCCAC16                                                            (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     CACAACAAGCTTCCACC17                                                           (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     CACACCAAACTTCCACCC18                                                          (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     CACATCAAGCTTCCACC17                                                           (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     CACAACAAGCTTCCACCC18                                                          (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     TACACAACAAGCTTCCACCC20                                                        (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     TACACACAAAGCTTCCACCC20                                                        (2) INFORMATION FOR SEQ ID NO: 17:                                             (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     TACACACCAGGCTTCCACCC20                                                        ( 2) INFORMATION FOR SEQ ID NO: 18:                                           (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     CACACCCAGCTTCCAC 16                                                           (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     TACACACCCAGCTTCCA 17                                                          (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     CACACCCAGCTT CCACC17                                                          (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     TACAC ACCCAGCTTCCAC18                                                         (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                      ACACCCAGCTTCCACC16                                                           (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     CACACCAAGATTCCAC16                                                            (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                    CACACCAAGATTCCACC17                                                           (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                    TACACACCAAGATTCCA17                                                           (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     AACACACCAAGATTCCA17                                                           (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: Other nucleic acid                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     TACACACCAAGATTCCAC18                                                          (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     GTGGAATCTTGGTGTGT17                                                           (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                              (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     GGTGGAATCTTGGTGTG17                                                           (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                              (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     GGTGGAATCTTGGTGTGT18                                                          (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     AGTGACTAGTGCTGCAAGAA20                                                        (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single stranded                                            (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     GGCCAGTGACTAGTGCTGCAAGAA24                                                    (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     ( B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     TGAAAGCTCTGCATCATGGGCAGTGA26                                                  (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     GGACAGGGCACTGGCCACTC20                                                        (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     AGACAATAAAGATGAACCCATAGTGAGC28                                                (2) INFORMATION FOR SEQ ID NO: 36:                                             (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     AGTGCCAACCGCCTCACAGG20                                                        (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     CCTCTCACACCAGTTCACTC20                                                        (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     GTGCTGCAGGTGTAAACTTGTAC CAG26                                                 (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     CACGGAT CCGGTAGCAGCGGTAGAGTTG28                                               (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     GGATGTGAGGAATTTGTCTTTTGCA25                                                   (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                    CCATTTGTCTGTGATGAGATGTAAC25                                                   (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: Other nucleic acid                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     CTAGGGATGTTCCTGTCTCAG21                                                       (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                              (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     TGCCAAGCCCTGTTCTGCGA20                                                        (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                              (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     CTGGCAGAGCGACTAAAAGCAAAATTG27                                                 (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single stranded                                            (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     ATCAATCTCTGAATCACAGTAAAGAGGAGG30                                              (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     AGGATATGGTCCTCTTCCAC20                                                        (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     TGGAAGAGAACCATATCCT19                                                         (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     CATTGCCACACCAGTGGTA19                                                         (2) INFORMATION FOR SEQ ID NO: 49:                                            (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                     TACCACTGAGGTGGCAATG19                                                         (2) INFORMATION FOR SEQ ID NO: 50:                                             (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:                                     CTTTCCCGGAATGCTG16                                                            (2) INFORMATION FOR SEQ ID NO: 51:                                             (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:                                     CAGCATTCCAGGAAAGG17                                                           (2) INFORMATION FOR SEQ ID NO: 52:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:                                     GATGACACACCCAAGG16                                                            (2) INFORMATION FOR SEQ ID NO: 53:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:                                     GATGCCAAACCCACGG 16                                                           (2) INFORMATION FOR SEQ ID NO: 54:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:                                     GGCCGTGGGGGTGGCCT C18                                                         (2) INFORMATION FOR SEQ ID NO: 55:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:                                     TTCTACGTG GACCTGGAGAGGAAGGAGACTGCCTG35                                        (2) INFORMATION FOR SEQ ID NO: 56:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                    AAGCTTGGTGTGTAG15                                                             (2) INFORMATION FOR SEQ ID NO: 57:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:                                    AAGCTGGGTGTGTAG15                                                             (2) INFORMATION FOR SEQ ID NO: 58:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:                                     AATCTTGGTGTGTAA15                                                         

We claim:
 1. An isolated sequence-specific oligonucleotide probe fordetecting the presence of a C allele of the Gγ-globin locus in a samplecontaining Gγ-globin locus nucleic acids, said sequence-specificoligonucleotide probe consisting essentially of a nucleotide sequence atleast 10 and less than 31 nucleotides in length which probe sequence isexactly complementary to said C allele.
 2. A panel of at least twosequence-specific oligonucleotide probes for determining an individual'sGγ-globin locus genotype from a sample containing Gγ-globin locusnucleic acid obtained from said individual said panel comprising atleast an isolated sequence-specific oligonucleotide probe consistingessentially of a hybridizing region at least 10 and less than 31nucleotides in length which hybridizing region is exactly complementaryto the C allele and an isolated sequence-specific oligonucleotide probeconsisting essentially of a hybridizing region at least 10 and less than31 nucleotides in length which hybridizing region is exactlycomplementary to either the A allele or the B allele.
 3. The panel ofclaim 2, wherein the hybridization region of each of saidsequence-specific oligonucleotide probes is a sequence of between 12 and24 nucleotides.
 4. The panel of claim 2, wherein said panel comprises ansequence-specific oligonucleotide probe with the hybridization regionSEQ ID NO: 1, and an sequence-specific oligonucleotide probe with thehybridization region SEQ ID NO: 18, and an sequence-specificoligonucleotide probe with the hybridization region SEQ ID NO:
 23. 5. Apanel of at least two sequence-specific oligonucleotide probes fordetermining an individual's Gγ-globin locus genotype from a samplecontaining Gγ-globin locus nucleic acid obtained from said individualsaid panel comprising isolated sequence-specific oligonucleotide probeseach probe consisting essentially of a hybridization region at least 10and less than 31 nucleotides in length which hybridization region isexactly complementary to the A, B and/or C allele.
 6. A kit useful fordetermining the genotype of an individual at the ^(G) γ-globin locuscomprising the panel of claim
 2. 7. The kit of claim 6, furthercomprising oligonucleotide primers useful for amplifying ^(G) γ-globinlocus nucleic acid.
 8. The kit of claim 7, wherein said panel ofsequence-specific oligonucleotide probes comprises an sequence-specificoligonucleotide probe with hybridization region SEQ ID NO: 1, ansequence-specific oligonucleotide probe with hybridization region SEQ IDNO: 18, and an sequence-specific oligonucleotide probe withhybridization region SEQ ID NO: 23, and containing a primer withhybridization region SEQ ID NO: 32 and a primer with hybridizationregion SEQ ID NO: 33.